ET_A/ET_A/B receptor antagonists increased coronary blood flow or dilated the coronary vasculature, depending on the species (Table 1). In dog, although intracoronarily (Thorin et al., 1999) and intravenously (Takamura et al., 2000) infused ET_A and ET_A/B receptor antagonists did not increase coronary flow (Table 1), infusion dilated the external coronary circumflex artery (Thorin et al., 1999) and increased coronary sinus O₂ saturation, consistent with coronary dilation (Takamura et al., 2000). ET_B receptor antagonism and NO synthase inhibition did not alter the diameter of the external coronary circumflex artery, although coronary flow decreased (Thorin et al., 1999).

In pig, intrapulmonary artery infused ET_A and ET_A/B receptor antagonist increased flow/dilated the coronary vascular bed (Merkus et al., 2002, 2003, 2005, 2006; de Beer et al., 2011b; Zhou et al., 2014a; Table 1). The magnitude of increase was slightly, but significantly greater with ET_A receptor antagonism as compared to ET_A/B receptor antagonism, suggesting the presence of underlying endothelial ET_B receptor vasodilation (Merkus et al., 2003; Table 1). Along these lines, although NO synthase and cyclooxygenase inhibition did not enhance ET_A/B receptor antagonist increased flow, it was speculated that ischemia due to NO synthase and cyclooxygenase inhibition resulted in release of a vasodilator, e.g., adenosine, which masked the decreased flow (Merkus et al., 2006). Indeed, the possibility that ET_A/B receptor antagonist inhibited NO release is supported by the lack of additivity of the elevated flows by ET_A/B receptor antagonist and an inhibitor of phosphodiesterase-5; the latter preventing the breakdown of the NO-mediator of vasodilation, cyclic GMP (Zhou et al., 2014a). Thus, even at rest, the capacity of the endothelium to synthesize/release ET-1 is reduced by NO. It should be noted, however, that an increase in ET-1 plasma levels in the coronary vascular bed (arteriovenous difference) was not detected following NO synthase inhibition (Merkus et al., 2006).

While an explanation is not clear with respect to the enhancement and lack of effect of ET_A/ET_A/B receptor antagonism on resting flow in the coronary vasculature of the pig (Merkus et al., 2002, 2003, 2005, 2006) and dog (Thorin et al., 1999; Takamura et al., 2000), respectively, one possibility is differences in the regulation of the coronary vasculature in these species (Merkus et al., 2006, 2008). In this regard, in dog coronary vasculature, cyclooxygenase and NO synthase inhibition was without effect and caused minimal constriction, respectively (as cited in Merkus et al., 2006, 2008). In contrast, in the coronary vasculature of the pig and, notably, in human, cyclooxygenase and NO synthase inhibition caused vasoconstriction (Merkus et al., 2006, 2008).

ET_A/ET_A/B receptor antagonists increased coronary blood flow and/or dilated the coronary vasculature, depending on the species (Table 1). In dog, intravenous infused ET_A/B receptor antagonist increased coronary sinus O₂ saturation (Takamura et al., 2000), consistent with coronary dilation, although elevated coronary flow was not detected (Takamura et al., 2000; Table 1). With ventricular pacing, which did not increase coronary flow, ET_A and ET_A/B receptor antagonists increased the diameter of the external coronary circumflex artery to a similar magnitude as observed with these antagonists at rest (Thorin et al., 1999). NO synthase inhibitor did not alter the ET_A and ET_A/B receptor antagonist-increased diameter of the external coronary circumflex artery with ventricular pacing (Thorin et al., 1999).

In pig, intrapulmonary artery infused ET_A and ET_A/B receptor antagonist increased coronary dilation, with the magnitude of increase slightly but significantly greater with ET_A receptor antagonist at all levels of exercise intensity (Merkus et al., 2002, 2003, 2005, 2006; de Beer et al., 2011b; Zhou et al., 2014a; Table 1). With increasing exercise intensity, however, the additional vasodilator effect due to ET_A and ET_A/B receptor antagonism waned and was obliterated, respectively (Merkus et al., 2003, 2005; Table 1). As with rest, the magnitude of increase was slightly but significantly greater with ET_A receptor antagonism as compared to ET_A/B receptor antagonism (Merkus et al., 2003; Table 1). Contrary to the coronary ET_A receptor vasoconstrictor influence that waned with increasing exercise intensity, the ET_B receptor mediation vasodilation was similar at all exercise intensities (Merkus et al., 2003, 2006).

Despite the underlying ET_B receptor mediated vasodilation, neither NO synthase inhibition nor cyclooxygenase inhibition enhanced the vasodilation by ET_A and ET_A/B receptor antagonism (Merkus et al., 2006). However, in the presence of inhibitors of NO synthase and/or cyclooxygenase, the vasodilator effect of ET_A/B receptor blockade was sustained with increasing exercise intensity, with combined inhibition eliciting additive effects (Merkus et al., 2006). Moreover, in the presence of NO synthase and cyclooxygenase inhibitors there was a positive correlation between ET_A/B receptor antagonist-increased flow and exercise intensity (Merkus et al., 2006; de Beer et al., 2011b). Thus, increased exercise intensity resulted in greater NO- and prostaglandin-mediated suppression of ET-1-mediated coronary vasoconstriction (Merkus et al., 2006).

Several findings suggest that the ability of NO synthase inhibitor to prevent the ET_A/B receptor antagonist obliteration of increased flow is due to reversal of NO inhibition of ET-1 synthesis. Increasing exercise intensity caused a dose-dependent decrease in ET-1 synthesis in the coronary vasculature, but not in the systemic circulation, as assessed by the conversion of big ET-1 to ET-1 (de Beer et al., 2011a). NO synthase inhibition also increased ET-1 plasma levels in the coronary vasculature (coronary arteriovenous difference; Merkus et al., 2006). Decreased ET-1 synthesis with exercise is also consistent with lowered pre-pro ET-1 mRNA expression in the aorta following 30 min (shortest duration studied) in the rat (Maeda et al., 2004). Further, the NO-mediated vasodilation was not due to decreased ET-1 constriction because contractile sensitivity to infused exogenous ET-1 remained unaltered with exercise (de Beer et al., 2011a). Also, phosphodiesterase-5 inhibition and 8-Br-cGMP did not decrease ET-1 constriction, whereas the endothelium-dependent constriction to big ET-1 was decreased in isolated coronary resistance vessels (Zhou et al., 2014a). These findings are consistent with NO synthase inhibitor-increased big ET-1, but not ET-1, constriction of isolated rat mesenteric artery (Bourque et al., 2012).

The ability of ET_A and ET_A/B receptor antagonists to increase coronary flow at rest and with exercise demonstrates that ET-1 dynamically, i.e., on a moment-to-moment basis, limits coronary flow under both conditions (Table 1). Moreover, the coronary endothelium has a greater capacity to release ET-1 with increased exercise intensity, an effect that is limited by NO inhibition of ET-1 synthesis. Thus, the dynamic regulation of flow by ET-1 is modulated by a dynamic balance between increased ET-1 synthesis and subsequent ET-1 release, and increased inhibition of ET-1 synthesis by enhanced NO.

It is of interest to consider that a major influence of the balance between NO and ET-1 is pulsatile shear stress. In this regard, the coronary bed is exposed to relatively high levels of shear stress even at rest (Recchia et al., 1996). Indeed, the magnitude of basal flow in the coronary vascular bed is similar to flow in exercised skeletal muscle (Laughlin et al., 1996). With increased exercise intensity, shear stress increases, resulting in further NO release and lowering ET-1 synthesis (Merkus et al., 2003, 2006; Rapoport, 2014b). Furthermore, it should be considered that along with a major effect of shear stress to increase NO release, endothelial ET_B receptor activation provides a relatively smaller component of NO, in particular at higher levels of exercise intensity.

Intra-aortic infused ET_A and ET_A/B receptor antagonists did not alter resting flow in intestine, kidney, spleen, and stomach (Maeda et al., 2002; Table 1).

Exercise decreased flow in intestine, kidney, spleen, and stomach (Maeda et al., 2002; Table 1). The amount of exercise-lowered flow was reduced by ET_A and ET_A/B receptor antagonism (Maeda et al., 2002; Table 1). Similar magnitudes of attenuation of the decreased flow with exercise were achieved with ET_A and ET_A/B receptor antagonists (Maeda et al., 2002; Table 1).

Consistent with ET-1 as a dynamic regulator of vascular tone, is the ET_A and ET_A/B receptor antagonist reduction of exercise-induced decreased flow to intestine, kidney, spleen, and stomach (Maeda et al., 2002; Table 1). Thus, ET-1 appears to regulate exercise-mediated redistribution of blood flow away from the splanchnic circulation to other organs, e.g., skeletal muscle (see below; Maeda et al., 2002). The similar magnitude of ET_A and ET_A/B receptor antagonist-mediated reduction of exercise-induced decreased flow (Maeda et al., 2002; Table 1) suggests that ET-1 activation of endothelial ET_B receptors does not modulate this reduction.

The lack of effect of ET_A and ET_A/B receptor antagonism on resting flow in splanchnic organs is consistent with the lesser magnitude of sheer stress to which these organs are subjected at rest as compared to the coronary vascular bed, in which ET_A and ET_A/B receptor antagonism increased resting flow (see section “Heart”; Table 1). That is, ET-1 release is increased with modest elevations in shear stress and the level of NO release may be insufficient to counter, through inhibition of ET-1 synthesis, the increased ET-1 release (Rapoport, 2014b).

ET_A and ET_A/B receptor antagonism were largely without effect on resting skeletal muscle blood flow in healthy human volunteers, as demonstrated in the forearm and leg with intra-brachial artery (Cardillo et al., 1999, 2000, 2002, 2004; Shemyakin et al., 2006; Van Guilder et al., 2007; Weil et al., 2011; Schreuder et al., 2014; Dow et al., 2017) and intra-femoral artery infused antagonist, respectively (Thijssen et al., 2007; Barrett-O’Keefe et al., 2013, 2015; Table 2). In contrast, other studies demonstrated increased forearm flow with intra-brachial ET_A and ET_A/B receptor antagonist infusion (Verhaar et al., 1998; Spratt et al., 2001; Martin et al., 2002; McEniery et al., 2002; Table 2).

An explanation for the contrasting effects of ET_A and ET_A/B receptor antagonists on resting flow does not appear to be the age of the volunteers. Specifically, while ET_A/ET_A/B receptor antagonist-increased flow in forearm and leg was restricted to aged humans (Van Guilder et al., 2007; Barrett-O’Keefe et al., 2013, 2015; Table 2), others demonstrated increased forearm flow with ET_A/ET_A/B receptor antagonism in relatively younger volunteers (Verhaar et al., 1998; Spratt et al., 2001). Another possibility is that undetected endothelial dysregulation is present in otherwise healthy volunteers. Along these lines, ET_A/ET_A/B receptor antagonism increased forearm resting flow in volunteers with hypercholesterolemia, insulin resistance, type 2 diabetes, excessive weight, and combined excessive weight and hypertension, but not in normal control volunteers (Cardillo et al., 2000, 2002, 2004; Mather et al., 2002; Shemyakin et al., 2006; Weil et al., 2011; Dow et al., 2017; Table 2). Possibly in general support of this explanation is that intra-aorta infused ET_A and ET_A/B receptor antagonists did not lower blood flow in plantaris, soleus, and tibialis muscle of rats 10 weeks of age (Maeda et al., 2002; Table 1). It should also be noted, though, that other studies were unable to demonstrate increased forearm flow with ET_A/ET_A/B receptor antagonism in volunteers with excessive weight and type 2 diabetes (Cardillo et al., 2004; Schreuder et al., 2014; Table 2).

In any case, the increased forearm flow at rest elicited by ET_A receptor antagonism was attributed to underlying endothelial ET_B receptor-mediated dilation because NO synthase inhibition and ET_B receptor antagonism reduced the ET_A receptor antagonist-induced increase in flow, and ET_B receptor antagonism alone decreased flow (Verhaar et al., 1998; Table 2). Along the general line of the involvement of NO-mitigation of the decreased flow due to ET-1, in volunteers with hypercholesterolemia and insulin resistance, ET_A, but not ET_A/B receptor antagonism, increased flow (Cardillo et al., 2000; Shemyakin et al., 2006). Although, in volunteers with type 2 diabetes and aged volunteers, ET_A and ET_A/B receptor antagonism elicited similar increases in flow (Cardillo et al., 2002; Van Guilder et al., 2007). Thus, NO-mitigation of decreased flow due to ET-1 appears to depend on the disease state.

In healthy human volunteers, exercise caused a similar magnitude of ET_A receptor antagonist-induced elevation of forearm blood flow as at rest (McEniery et al., 2002; Table 2). In the leg, in contrast to the lack of ET_A receptor antagonist-increased flow at rest, ET_A receptor antagonism increased flow with exercise (Barrett-O’Keefe et al., 2013, 2015). Hypertension, type 2 diabetes, and age were associated with ET_A/ET_A/B receptor antagonist increased flow in forearm and leg with exercise and were even enhanced as compared to the ET_A receptor antagonism increased flow observed at rest (McEniery et al., 2002; Barrett-O’Keefe et al., 2013, 2015; Schreuder et al., 2014; Table 2). Although plethysmography-induced reactive hyperemia was increased by ET_A/B receptor antagonism to similar magnitudes in healthy and hypertensive volunteers (Martin et al., 2002; Table 2), it is not clear whether plethysmography-induced reactive hyperemia mimics the effects exercise.

In rat, intra-aortic infusion of ET_A and ET_A/B receptor antagonists actually resulted in a reduction in flow in tibialis and plantaris muscle with exercise as compared to rest (Maeda et al., 2002; Table 1). It should be considered, however, that the reduced flow was a direct effect of the decreased arterial pressure due to intra-aortic infusion of ET_A and ET_A/B receptor antagonist (Maeda et al., 2002).

The involvement of ET-1 production in exercise is equivocal. With one-legged exercise, ET-1 production increased in the non-exercising leg, whereas it was not changed in the exercising leg (Maeda et al., 1997). Conversely, ET-1 production was increased during knee exercise-extensor exercise in the exercising leg (Barrett-O’Keefe et al., 2013). An additional complexity is that NO formation, which is increased with exercise as demonstrated through NO synthase inhibition of dilation of the brachial artery with handgrip (Wray et al., 2011), would be anticipated to decrease ET-1 production increased by pulsatile shear stress (Rapoport, 2014b).

Changes in ET-1 contractile sensitivity of the skeletal muscle vasculature also occur with exercise. Indeed, the vasoconstrictor influence of infused ET-1 was progressively reduced with incremental exercise intensity in the leg (Wray et al., 2007).

These findings (Table 2) are consistent with ET-1 as a dynamic regulator of vascular tone in the skeletal muscle with exercise. The changes in production and sensitivity together result in an altered contribution of ET-1 to skeletal muscle blood flow during exercise. The variable effects of ET-1 involvement at rest may point to different degrees of underlying endothelial dysregulation. Indeed, pathophysiologies associated with endothelial dysregulation also demonstrate dynamic ET-1 dependency of flow with rest and exercise.

In lying and standing young pig (2–3 months), an intra-pulmonary artery infused ET_A receptor antagonist was without effect on pulmonary arterial pressure (Merkus et al., 2003, 2007; Houweling et al., 2006; de Beer et al., 2010; Zhou et al., 2014b; Table 1). While ET_A/B receptor antagonism also did not lower pulmonary arterial pressure in lying pig, in a number of studies pressure was lowered in standing pig (Merkus et al., 2003, 2007; Houweling et al., 2006; de Beer et al., 2010; Zhou et al., 2014b; Table 1). The lack of role of ET-1 in basal tone in lying pig may be due to ongoing NO inhibition of ET-1 synthesis because NO synthase inhibition increased pulmonary vascular resistance and an ET_A/B receptor antagonist prevented the increase (Merkus et al., 2007). Further, in standing pig (not investigated in lying pig), pulmonary arterial pressure was reduced by phosphodiesterase-5 inhibition and ET_A/B receptor blockade failed to further reduce pressure in the presence of phosphodiesterase-5 inhibition (Zhou et al., 2014b).

ET_A/B, but not ET_A receptor antagonism, prevented exercise-induced increased pulmonary arterial pressure (Merkus et al., 2003; Table 1). Thus, it was concluded that the ET-1-elevated tone during exercise was mediated by (smooth muscle) ET_B receptors (Merkus et al., 2003). Indeed, ET_B receptor-mediated, ET-1-elevated tone of the pulmonary vasculature during exercise is consistent with ET_B receptor mediation of constriction of pulmonary resistance vessels (MacLean et al., 1994).

Also, ongoing NO release inhibits the ET-1 increase in resistance as demonstrated by enhancement of the ET_A/B receptor antagonism-induced decrease in pulmonary arterial pressure with NO synthase inhibition (Merkus et al., 2007). Consistent with an underlying role for NO in inhibition of ET-1 release is lack of further increase in pulmonary arterial pressure by ET_A/B receptor following inhibition of phosphodiesterase-5 (Zhou et al., 2014b). Furthermore, the effect of phosphodiesterase-5 inhibition to lower pulmonary arterial pressure was due to decreased ET-1 synthesis, as demonstrated by inhibition of big ET-1 constriction in isolated pulmonary resistance vessels (Zhou et al., 2014b).

Consistent with ET-1 as a dynamic regulator of vascular tone is the ET_A/B receptor antagonist reduction of exercise-induced decreased pulmonary arterial pressure by ET_A/B receptor antagonism (Table 1). The release of ET-1 in response to exercise is consistent with the ability of moderate levels of pulsatile shear stress to increase ET-1 release (Rapoport, 2014b).

The lack of effect of ET_A/B receptor antagonism on resting pressure (lying pig) presumably reflects in part lower ET-1 release due to lesser pulsatile shear stress, i.e., similar to that observed in splanchnic organs (see section “Splanchnic: Intestine, Kidney, Spleen, and Stomach”). Another factor involved in the lack of effect of ET_A/B receptor antagonism at rest is NO inhibition of ET-1 synthesis. While the relative contribution of NO released in response to endothelial ET_B receptor activation and shear stress is not clear, it may be reasonable to consider that the relative amount of NO release by endothelial ET_B receptor activation is considerably greater than in the coronary vasculature (see section “Heart”).